Liquid crystalline substrates for culturing cells

ABSTRACT

The present invention is directed to liquid crystalline substrates useful in the culture of cells and methods of their use. In certain embodiments, the invention provides methods and devices for imaging changes (e.g., reorganization) of extracellular matrix components by living cells.

CROSS REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. application Ser. No.12/777,320, filed on May 11, 2010 and issued as U.S. Pat. No. 7,947,500on May 24, 2011, which is a divisional of U.S. application Ser. No.11/565,363, filed on Nov. 30, 2006 and issued as U.S. Pat. No. 7,732,152on Jun. 8, 2010, which claims the benefit of U.S. ProvisionalApplication No. 60/740,944, filed Nov. 30, 2005. All of theseapplications are incorporated by reference herein in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under DMR-0079983awarded by the National Science Foundation. The government has certainrights in the invention.

FIELD OF THE INVENTION

This invention relates generally to liquid crystal technology. Moreparticularly, the present invention is directed to liquid crystallinesubstrates for culturing cells and related methods.

BACKGROUND OF THE INVENTION

The functions of cells in biological systems are guided by the spatialand temporal delivery of a range of chemical and mechanical cues. A widevariety of engineered materials have been investigated with the goal ofexerting control over cell behaviors such as adhesion to surfaces,differentiation, and proliferation in in vitro and biomedicalapplications. The interactions of cells with solid surfaces, forexample, have been engineered by controlling surface chemistry,topographical features, compliance of the substrate, and combinations ofthese parameters. Synthetic materials have also formed the basis ofapproaches that measure the interactions between cells and theirenvironments. For example, certain approaches have previously beendescribed that use microfabricated elastomeric posts to measure thepatterns of mechanical forces exerted by cells on surfaces.

The liquid crystalline state is widely encountered in biologicalsystems. For example, the membranes of cells are liquid crystalline, andconcentrated solutions of biomolecules such as DNA and proteins areknown to form liquid crystals. In light of this, it is surprising thatfew studies aimed at engineering synthetic materials to interact withlive cells have explored the use of synthetic liquid crystals. Paststudies have investigated the orientations of the nematic liquid crystal4′-pentyl-4-cyanobiphenyl (5CB) on cells attached to surfaces. Thesecells, however, were fixed (i.e., dead) and the liquid crystal used intheir investigation (5CB) has subsequently been shown to cause celldeath when contacted with live cells for short periods of time. In aseparate study, Luk and coworkers surveyed liquid crystalline materialscontaining a variety of functional groups and found several liquidcrystals that were non-toxic to live mammalian cells. In particular,fluorinated liquid crystals exhibited minimal toxicity toward 3T3fibroblasts and corneal epithelial cells.

Accordingly, there exists a need in the field to engineer syntheticmaterials that, in a first aspect, offer a suitable culture substratefor living cells and, in a second aspect, provide means to monitor cellbehaviors such as adhesion to surfaces, differentiation, andproliferation.

SUMMARY OF THE INVENTION

In a first embodiment, the present invention is directed to methods forculturing cells on a cell culture substrate. Such methods include stepsof: (a) providing a cell culture substrate by: (i) preparing aninterface between an aqueous phase and a liquid crystalline phase; and(ii) depositing a protein or peptide layer at the interface; (b) seedingliving cells onto the protein or peptide layer; and (c) culturing theliving cells on the cell culture substrate.

In a preferred embodiment, the protein layer comprises at least oneprotein or peptide that is an extracellular matrix component and, inparticularly preferred embodiments, the extracellular matrix componentis collagen, gelatin, laminin, or elastin. Alternatively, or inaddition, the protein or peptide layer may comprise synthetic peptidesand proteins such as, e.g., polylysine.

In certain embodiments, the proteins and peptides are deposited at theinterface from cell culture media. In other embodiments, the proteins orpeptides of the protein layer are further incorporated intopolyelectrolyte multilayer films deposited at the interface.

The liquid crystal is substantially non-toxic to living cells and can bea low molecular weight liquid crystal, a polymeric liquid crystal, acomposite material containing a liquid crystal or a liquid crystallineelastomeric material.

The present invention is further directed to methods for detectingcell-induced changes to a cell culture substrate by living cells. Suchmethods include steps of: (a) providing a cell culture substrate by: (i)preparing an interface between an aqueous phase and a liquid crystallinephase; and (ii) depositing a protein or peptide layer at the interface;(b) seeding living cells onto the protein or peptide layer of the cellculture substrate; (c) maintaining the living cells under cultureconditions; and (d) detecting reorganization of the liquid crystallinephase. The reorganization indicates cell-induced changes between theliving cells and the cell culture substrate. In a particularly preferredembodiment, the living cells are human embryonic stem cells and themethod provides for visualizing developmental changes occurring in thecultured cells.

The invention also encompasses cell culture substrates that include: (a)an aqueous phase; (b) a liquid crystalline phase wherein an interface isformed between the aqueous phase and liquid crystalline phase; and (c) aprotein or peptide layer positioned at the interface. In preferredembodiments, the protein or peptide layer comprises at least one proteinor peptide that is an extracellular matrix component and, in preferredembodiments, the protein layer includes collagen, gelatin, laminin, orelastin. Alternatively, or in addition, the protein layer may comprise asynthetic polypeptide or a naturally occurring peptide or protein. Incertain embodiments, the proteins or peptides of the protein layer arefurther incorporated into a polyelectrolyte multilayer film deposited atthe interface. The liquid crystal is substantially non-toxic to livingcells and can be a low molecular weight liquid crystal, a polymericliquid crystal, a composite material containing a liquid crystal or aliquid crystalline elastomeric material.

In yet another aspect, the invention provides a device for detectingcell-induced changes to a cell culture substrate by living cells,comprising: (a) a cell culture substrate having: (i) an aqueous phase;(ii) a liquid crystalline phase wherein an interface is formed betweenthe aqueous phase and liquid crystalline phase; and (iii) a protein orpeptide layer positioned at the interface; and (b) a means for imagingreorganization of the liquid crystalline phase. Reorganization indicateschanges to the cell culture substrate induced by cells cultured on thecell culture substrate.

Other objects, features and advantages of the present invention willbecome apparent after review of the specification, claims and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. A) Schematic illustration of the geometry for producinginterfaces between aqueous phases and immiscible thermotropic liquidcrystals. Note that the thicknesses of the slide, grid, and Matrigelfilm are not to scale. The Matrigel thickness indicated is based on thethickness of a dried Matrigel film on glass (˜10 nm). Dashed lines inthe TL205 liquid crystalline film represent an example of the directorprofile for planar anchoring. B) The liquid crystal TL205 is aproprietary mixture of cyclohexane-fluorinated biphenyls and fluorinatedterphenyls; representative chemical structures are shown. C)Fluorescence image of TL205 following exposure to a FITC-labeledMatrigel solution for 2 h under conditions leading to gelation. D-F)Optical images (crossed polars) of TL205 after 2 h in contact withphosphate buffer (D), a solution of Matrigel under conditions leading togelation (E), or a 1 μM solution of bovine serum albumin (F). Scale barsare 100 μm.

FIG. 2. Phase-contrast (A-D), polarized (E-H), and fluorescence (I-L)images of human embryonic stem cells seeded on a Matrigel-coated TL205substrate and imaged after the times shown for each column. Scale barsare 300 μm for A, E, and I; all others are 100 μm.

FIG. 3. A-B) Optical images (crossed polars) of TL205 in contact withcell medium for four days with (A) and without (B) a layer of Matrigelat the aqueous-TL205 interface. C) Optical image (crossed polars) ofTL205 after 3 h in contact with a 100 μM suspension of 2DLPC liposomesin PBS. D) Optical image (crossed polars) of TL205 after 30 min incontact with a mixture of 100 μM DLPC and 1 μM BSA in PBS. Scale barsare 100 μm.

FIG. 4. DiD-labeled 3T3 cells on polyethylenimine/(hyaluronicacid/collagen)₅-TL205. Pictures were acquired from different wells.Images were taken 10 hours post-cell seeding.

FIG. 5. NIH 3T3 cell growth on OTS-coated glass (left panel, phasecontrast image) and LCE (right panel, fluorescent image of Di-D treatedcells) in a well treated with bovine calf serum. Images were taken 12hours post-cell seeding.

FIG. 6 NIH 3T3 cell growth on serum-treated LCE (the same sampledepicted in the right panel of FIG. 5). The image was taken on the5^(th) day post-cell seeding.

DETAILED DESCRIPTION OF THE INVENTION I IN GENERAL

Before the present materials and methods are described, it is understoodthat this invention is not limited to the particular methodology,protocols, materials, and reagents described, as these may vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto limit the scope of the present invention which will be limited onlyby the appended claims.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural reference unless thecontext clearly dictates otherwise. As well, the terms “a” (or “an”),“one or more” and “at least one” can be used interchangeably herein. Itis also to be noted that the terms “comprising”, “including”, and“having” can be used interchangeably.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications and patentsspecifically mentioned herein are incorporated by reference for allpurposes including describing and disclosing the chemicals, instruments,statistical analysis and methodologies which are reported in thepublications which might be used in connection with the invention. Allreferences cited in this specification are to be taken as indicative ofthe level of skill in the art. Nothing herein is to be construed as anadmission that the invention is not entitled to antedate such disclosureby virtue of prior invention.

II. THE INVENTION

Synthetic liquid crystals possess a number of properties that may makethem useful when engineering interfaces to live cells. First, liquidcrystals are anisotropic elastic fluids with splay, bend and twistelastic constants often on the order of 1 pN. The values of theseelastic constants can be manipulated substantially by changes in thestructure of the molecules comprising the liquid crystals. Consequently,these materials can reorganize under the influence of stressescomparable in magnitude to those transmitted from cells to theirenvironments, thus potential providing a facile manner for reportingmechanical interaction of cells and their environments. Theorientational order of liquid crystals near interfaces (the “anchoring”of the liquid crystal) is known to be highly sensitive to the nature ofthe interactions between the mesogens forming the liquid crystal and aconfining interface. For example, the orientation of liquid crystals hasbeen shown to be coupled to the presence and organization ofphospholipids and proteins adsorbed at aqueous-liquid crystalinterfaces. The orientation of liquid crystals are also dependent on thechemical structure of interfaces that they contact and thus changes inchemical structure or organization of molecules induced for example byenzymes that are secreted by cells can also be reported by liquidcrystals.

The present invention provides liquid crystal-based substrates on whichcells may be cultured. Such substrates may be incorporated into devicesthat measure and detect cell-induced changes in a cell culture substrate(e.g., a protein layer) by living cells cultured on the substrate. Suchmethods involve the deposition of a thin film of a peptide or proteinlayer (e.g., Matrigel) on the surface of a thermotropic liquid crystal,and the seeding of the cells on the decorated liquid crystal. It ispossible to use the crystals to image the mechanical reorganization ofprotein or peptides by living cells. As well, the present inventionprovides certain methods and devices that permit facile observation ofthe reorganization of the extracellular matrix of cells. The inventionalso permits measurement of the mechanical forces transmitted by cellsto surfaces.

Accordingly, in a first aspect the invention provides methods forculturing cells on a liquid crystalline-based cell culture substrate.Such methods include steps of: (a) providing a cell culture substrateby: (i) preparing an interface between an aqueous phase and a liquidcrystalline phase; and (ii) depositing a protein or peptide layer at theinterface; (b) seeding living cells onto the protein or peptide layer;and (c) culturing the living cells on the cell culture substrate.

The protein layer preferably includes at least one protein or peptidethat is or may function as an extracellular matrix component such as,but not limited to, gelatin, laminin or collagen. The material availableunder the tradename Matrigel is a particularly preferred composition foruse as the protein layer. Matrigel has demonstrated particular utilityin this invention for the culture of human embryonic stem cells.However, the protein layer may, alternatively or in addition, comprise asynthetic polypeptide such as, e.g., polylysine.

One specific embodiment described herein utilizes an approximatelyplanar interface between the nematic liquid crystal TL205 (a proprietarymixture of cyclohexane-fluorinated biphenyls and fluorinated terphenyls)and an aqueous solution or cell media (FIG. 1A-B). The use of theapproximately planar interface is not a limitation of the invention, andthe invention includes interfaces that are not planar. In particular,the invention includes droplets of liquid crystals that will form curvedinterfaces with aqueous phases. In the described example, theapproximately planar interfaces were prepared by hosting the TL205 in agold TEM grid, which stabilizes the aqueous-TL205 interface. Thisgeometry permits easy observation and interpretation of the orientationsof the liquid crystals at the aqueous-TL205 interface and permitsexchange of the aqueous phase or cell media.

The term “liquid crystal”, as used herein, refers to an organiccomposition in an intermediate or mesomorphic state between solid andliquid. Suitable liquid crystals for use in the present inventioninclude, but are not limited to, thermotropic liquid crystals that aresubstantially nontoxic to living cells. The term “substantiallynon-toxic” refers to compositions that exhibit little or no toxicitywith respect to living cells, particularly eukaryotic cells, and mostpreferably mammal cells. Such compositions include liquid crystals withchemical functional groups such as fluorine atoms, fluorophenyl groups,or difluorophenyl groups. Preferred such liquid crystals include Cseries liquid crystals, TL205 and cholesterics, and substantiallynon-toxic liquid crystals can be identified by measurements ofcytotoxicity, using the methods and criteria reported by Luk andcoworkers in Luk, Y. Y.; Campbell, S. F.; Abbott, N. L.; Murphy, C. J.“Non-Toxic Thermotropic Liquid Crystals for Use with Mammalian Cells”,Liquid Crystals, 31, 611 (2004).

The invention is not limited to the non-toxic liquid crystalsspecifically identified by Luk and coworkers. It may employ polymericliquid crystals, composite materials comprising particles and liquidcrystals, or polymers and liquid crystals, as well as elastomeric liquidcrystals. A preferred liquid crystalline elastomer is synthesized fromthe mesogen M4OCH3 and polymethylhydrosiloxane, according to A. Komp andcoworkers “A versatile preparation route for thin free standing liquidsingle crystal elastomers” Macromol. Rapid Commun, 26: 813-818, 2005.Other LC elastomers suitable for use in the current invention aredescribed by Deng in “Advances in liquid crystal elastomers”, Progressin Chemistry, 18 (10): 1352-1360, 2006, and references cited therein.The scope of the invention also includes use of liquid crystallinehydrogels, as described by Weiss, F. and Finkelmann H. inMacromolecules; 37(17); 6587-6595, 2004, and references cited therein.Other embodiments use a composite comprising a dispersion of solidparticulates, such as but not limited to microspheres, mixed with liquidcrystal. Such composites are known by those skilled in the art to form agel.

Furthermore, the invention is not limited to the non-toxic liquidcrystals identified by Luk and coworkers. It is anticipated that in somecases, the presence of the protein substrate or other adsorbed/depositedlayer on the liquid crystal will lower the toxicity of liquid crystalsto cells. For example, plasma treatment of the surface of a liquidcrystalline elastomer can be used to lower the toxicity of the liquidcrystal.

A distinctive feature of this system is that the liquid crystal layer isin direct contact with the peptide or protein layer (e.g., thecomposition available under the tradename Matrigel), which in turn is incontact with the cells. As cells grow, differentiate, migrate, invadematrices, change shape, and undergo other changes of state, they impartsubtle structural changes to the protein or peptide layer. These changesare amplified and transduced by the liquid crystals, which become aneffective readout. Matrigel is the trade name for a gelatinous proteinmixture secreted by mouse tumor cells and currently available from BDBiosciences. This mixture resembles the complex extracellularenvironment found in many tissues and is used by cell biologists as asubstrate for cell culture. However, there are many alternatives toMatrigel for use in the present invention. Alternatives include otherproteins commonly found in the extracellular matrix of cells, includinglaminins, collagens, gelatin and other modified extracellular matrixproteins. The current invention also anticipates the use of synthetic orsemisynthetic peptide-containing layers where the peptide sequence leadsto interactions with the cell such as attachment of the cells to thelayers. These layers, protein and semisynthetic, can be deposited at theinterface of the liquid crystals using a variety of methods, includingphysical adsorption from solution, gelation and adsorption fromsolution, incorporation into multilayer structures formed at theinterface such as polyelectrolyte multilayer (PEM) films, incorporationinto amphiphilic molecules such as peptide amphiphiles, and covalentattachment to the liquid crystal. An example of a synthetic peptidesequence that can be used with the present invention is polylysine.

PEM films are particularly advantageous in that agents may beincorporated into their multilayer design such that the agent may bedelivered to the local environment of the cultured cells atpredetermined times and/or conditions. For example, a certain chemokinemay be incorporated into a degradable PEM film, cells cultured on thePEM coated LC substrate, and, upon degradation of the PEM, the chemokineis released and contacted with the cells. As can be appreciated, a widevariety of agents may be incorporated into PEM films in aspatially-desired manner, including, but are not limited to, drugs,ligands, chemokines, peptides, chemical functionalities, and nutrients.PEM films also provide a stiffer interface than the interface betweenculture medium and liquid crystal. This stiffness been shown to affectseveral cell phenotypes, including spreading, adhesion, motility, andproliferation.

Therefore, in alternative methods, a step of forming a polyelectrolytemultilayer (PEM) film at the interface between the aqueous phase andliquid crystalline phase is further included. Accordingly, the proteinlayer comprises the PEM film.

Although a wide variety of cells may be cultured according to thepresent invention, living human embryonic stem cells are a particularlypreferred cell type. Human embryonic stem (ES) cells, utilized in theillustrative example herein, are highly “plastic” and exceptionallysusceptible to cues from their environment. Human ES cell proliferationand differentiation have both been tied to signaling pathways that aretightly connected to the cell microenvironment. For example, previousstudies have shown that manipulation of the environment surroundinghuman ES cells by physical methods such as mechanical strain can lead tosuppression of differentiation pathways.

In addition to stem cell-related applications, the present invention hasutility for a variety of cell types, including corneal epithelial cells,fibroblasts, etc. The inventors have demonstrated the ability to growfibroblasts and human embryonic stem cells on a substrate according tothe invention. The inventors have passaged stem cells repeatedly andmaintained them in the undifferentiated state on this substrate.

In yet another aspect, the invention provides methods for detectingcell-induced changes to a cell culture substrate by living cells. Suchmethods includes steps of: (a) providing a cell culture substrate by:(i) preparing an interface between an aqueous phase and a liquidcrystalline phase; and (ii) depositing a protein or peptide layer at theinterface; (b) seeding living cells onto the protein or peptide layer ofthe cell culture substrate; (c) maintaining the living cells underculture conditions; and (d) detecting reorganization of the liquidcrystalline phase. The reorganization indicates cell-induced changesbetween the living cells and the cell culture substrate. In aparticularly preferred embodiment, the living cells are human embryonicstem cells and the method provides for visualizing developmental changesoccurring in the cultured cells.

In yet another aspect, the invention encompasses a liquid crystallineculture substrate upon which cells are cultured. Such a substrateincludes the elements of: (a) an aqueous phase; (b) a liquid crystallinephase wherein an interface is formed between the aqueous phase andliquid crystalline phase; and (c) a protein or peptide layer positionedat the interface. As previously-described, the protein or peptide layerpreferably includes at least one protein or peptide that is or mayfunction as an extracellular matrix component such as, but not limitedto, gelatin, laminin collagen, elastin, or a mixture thereof. Thematerial available under the tradename Matrigel is a particularlypreferred composition for use as the protein layer. Synthetic peptidesmay comprise the layer in alternative fashion or in addition toextracellular matrix components.

Although a broad variety of liquid crystalline substances may be used inpracticing the invention, certain preferred embodiments utilize a liquidcrystalline phase that is a liquid crystal elastomer. In certainsubstrates, a polyelectrolyte multilayer (PEM) film is positioned at theinterface between the aqueous phase and liquid crystalline phase.Accordingly, the protein layer is comprised by the PEM film.

Substrates according to the invention may further be incorporated intodevices for detecting cell-induced changes to a protein layer by livingcells. These devices include: (a) a cell culture substrate having: (i)an aqueous phase; (ii) a liquid crystalline phase wherein an interfaceis formed between the aqueous phase and liquid crystalline phase; and(iii) a protein or peptide layer positioned at the interface; and (b) ameans for imaging reorganization of the liquid crystalline phase.Reorganization indicates changes to the cell culture substrate inducedby cells cultured on the cell culture substrate.

As is known to those skilled in the art, changes in optical propertiesof the liquid crystal can be quantified by using optical instrumentationsuch as, but not limited to, plate readers, cameras, scanners,photomultiplier tubes. Because the dielectric properties of liquidcrystals also change with orientational order, measurements ofelectrical properties of liquid crystals can also be used to reportchanges in the interactions of molecules with liquid crystals. Thus awide range of optical and electrical methods for transduction ofcell-induced reorganization of liquid crystals is anticipated by thisinvention.

The following examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.Indeed, various modifications of the invention in addition to thoseshown and described herein will become apparent to those skilled in theart from the foregoing description and the following examples and fallwithin the scope of the appended claims.

III. EXAMPLES

The following materials and methodologies were utilized in the examplesdiscussed in greater detail below.

Materials. The nematic liquid crystal TL205 was purchased from EMDChemicals (Hawthorne, N.Y.) and used without further purification. Goldspecimen grids (bars 20 μm thick and 55 μm wide, spaced 283 μm apart)were obtained from Electron Microscopy Sciences (Fort Washington, Pa.).The phospholipid 1,2-dilauroyl-sn-glycero-3-phosphocholine was purchasedfrom Avanti Polar Lipids (Alabaster, Ala.) and used as received. Bovineserum albumin was purchased from Jackson ImmunoResearch Labs (WestGrove, Pa.). Deionization of a distilled water source was performed witha Milli-Q system (Millipore, Bedford, Mass.) to give water with aresistivity of 18.2 MΩ·cm. Glass microscope slides were Fisher's FinestPremium Grade obtained from Fisher Scientific (Pittsburgh, Pa.).Octadecyltrichlorosilane (OTS) was obtained from Fisher Scientific.8-well chamber slides used for cell culture were purchased from NalgeNunc International (Rochester, N.Y.). Matrigel was obtained fromInvitrogen (Carlsbad, Calif.) and labeled with the succinimidyl ester,Alexa Fluor 430 carboxylic acid from Molecular Probes (Carlsbad,Calif.). The human embryonic stem cell lines H1 and H9 were derived asdetailed elsewhere. J. Thomson, J. Itskovitz-Eldor, S. Shapiro, M.Waknitz, J. Swiergiel, V. Marshall, J. Jones, Science 1998, 282, 1145.ES cell medium contained 80% DMEM/F12 (Invitrogen), 20% knockout serumreplacer (Invitrogen), 1% L-glutamine, and 0.1 mM MEM non-essentialamino acids solution (Sigma-Aldrich, St. Louis, Mo.). ES cell medium wasconditioned on mouse embryonic fibroblasts (MEFs) and supplemented withbFGF (Invitrogen). MEF medium contained 90% DMEM (Invitrogen), 10% FBS,and 1% MEM nonessential amino acids solution.

Preparation of optical cells. A detailed description of the methods usedto prepare and examine the liquid crystal hosted within optical cellscan be found in a previous publication. J. M. Brake, N. L. Abbott,Langmuir 2002, 16, 6101. Briefly, glass microscope slides were cleanedaccording to published procedures and coated withoctadecyltrichlorosilane (OTS). The quality of the OTS layer wasassessed by checking the alignment of 5CB confined between twoOTS-treated glass slides. Any surface not causing homeotropic anchoringof 5CB was discarded. A small square of OTS-coated glass (ca. 5 mm×5 mm)was fixed to the bottom of each well of an 8-well chamber slide withepoxy and cured overnight at 60° C. The wells were rinsed several timeswith ethanol to remove uncured monomer and subsequently dried. Goldspecimen grids that were cleaned sequentially in methylene chloride,ethanol, and methanol were placed onto the surface of the OTS-treatedglass slides, one per well. Approximately 1 μL at of TL205 was dispensedonto each grid and the excess liquid crystal was removed with a syringeto produce an approximately planar interface.

Preparation of Fluorescently-labeled Matrigel. Matrigel was labeled withthe epifluorescent succinimidyl ester, Alexa Fluor 430 carboxylic acid.2 mg of frozen Matrigel was resuspended in 8 mL of 0.1 M sodiumbicarbonate buffer at 4° C. 0.2 mL of the Matrigel solution was added toeach well 8-well chamber slides containing TL205-impregnated grids orglass substrates. The slides were incubated at 37° C. for 2 h to allowformation of Matrigel ECM. After incubation, each well to be labeled waswashed twice with 0.4 mL sodium bicarbonate buffer. 0.2 mg of AlexaFluor 430 in 10 μL methanol was resuspended in 20 μL dimethyl sulfoxide.The resulting solution was added to 6.4 mL sodium bicarbonate buffer and200 μL was then transferred to each well. The chamber slides werecovered with foil to prevent photobleaching and incubated at roomtemperature for 1 h. Finally, the wells were washed 3 times in PBS toremove excess dye before cell culture.

Cell culture. Human ES cells were cultured on a Matrigel ECM using EScell medium conditioned on MEFs, supplemented with 4 ng/mL bFGF (CM/F+).Matrigel was deposited onto TL205 or glass substrates in chamber slidesas described above. ES cell colonies grown to passaging confluence(1-2×106 cells) were partially detached from their plate incubating 3-5min in 10 mg/mL dispase at 37° C. Colonies were washed once inunconditioned ES cell medium without bFGF (UM/F−) and then gentlyscraped from the plate using a glass pipette. ES cell colonies werecentrifuged 5 min at 1000 rpm in a Thermo IEC Centra CL2 tabletopcentrifuge and resuspended in 2.5 mL CM/F+. 0.3 mL of the resuspendedcells was then added to each well of the 8-well chamber slide containingTL205-impregnated grids or glass controls. The cell medium was changeddaily thereafter.

Cell viability assay. Live cells were detected with the LIVE/DEADViability/Toxicity Kit (Molecular Probes #L-3224) following themanufacturer's protocol. Briefly, ES cells that had been cultured onTL205 substrates were rinsed 3 times with PBS and then 1 μM calcein-AMin PBS was added to each well. After 20 min, samples fluorescencemicroscopy was used to characterize live cells, which showed up brightdue to the conversion of the non-fluorescent calcein-AM to fluorescentcalcein. Cell death was not characterized with the LIVE/DEAD kit due toconflicts between the stain used for dead cells and the fluorescent tagused in Oct-4 staining, which characterized cell differentiation.

Staining of the Oct-4 transcription factor. To visualize differentiationof ES cells, cells were stained for the transcription factor Oct-4,which is expressed solely in undifferentiated ES cells. First, cellswere fixed by incubating 15 min in 4% paraformaldehyde (200 mL/well) atroom temperature. Next, cells were blocked using 5% milk in PBS at 4° C.for 1 h. The primary antibody, mouse anti-Oct-3/4 (Santa CruzBiotechnology, Santa Cruz, Calif.), was diluted 1:200 in PBS+0.4% TritonX-100. 150 μL of this solution was added to each well and incubated 1 hat room temperature. Cells were then washed 5 times in PBS. Thesecondary antibody, Texas Red labeled goat anti-mouse (Molecular Probes)was diluted 1:500 in PBS+0.4% Triton. Cells were incubated in 150 μL ofthe secondary antibody solution for 1 h at room temperature. Finally,cells were washed 3 times and stored in PBS.

Flow cytometry and analysis. Following culture on TL205, cells werecollected from the liquid crystal interface. Cells on the liquid crystalgrids were isolated from those on the surrounding surface by placing arubber tube with diameter approximately equal to the grid diameter ontop of each grid. 0.05% trypsin/EDTA was injected into the center of thetubing and cells were incubated 10 min at 37° C., thereby dissociatingonly the cells growing on the TL205 and gold grid. Cells from four gridswere combined to a single centrifuge tube for analysis. Trypsin wassubsequently neutralized by the addition of 1 mL MEF medium, and thecells were centrifuged and washed once in FACS buffer (Ca/Mg2+-free PBS,2% FBS, 0.1% sodium azide). A mouse anti-SSEA-4 antibody (Santa CruzBiotechnology), which targets an ES cell marker, was diluted 1:50 inFACS buffer. Cells were resuspended in 100 μL of the primary mouseanti-SSEA-4 solution and incubated 30 min at room temperature. Cellswere washed twice in FACS buffer, and incubated 390 min at roomtemperature in a goat anti-mouse labeled phycoerythrin solution (diluted1:50 in FACS buffer). Samples were wrapped in foil to preventphotobleaching. Finally, samples were washed twice in FACS buffer andresuspended in a final volume of 300 μL FACS buffer for analysis. Datawere collected on a FACScan flow cytometer (Beckton Dickinson, FranklinLakes, N.J.) and analysis was performed on CellQuest (Beckton Dickinson)and WinMDI software. Cell viability was defined as whole cells (measuredvia light scattering) with intact membranes (measured as exclusion ofthe fluorescent live/dead marker propidium iodide). All whole cellsexcluding the live/dead marker were gated and SSEA-4 uptake was based onfluorescence levels of viable cells.

Liposome preparation. Chloroform solutions of pure DLPS or DLEPC weredispensed in a glass tube and dried under a low flow of N2 to form athin lipid film. Residual solvent was removed under vacuum at 50° C. forseveral hours. The resulting lipid film was hydrated overnight at roomtemperature (above the lipid transition temperature) with an appropriatevolume of water to yield a final lipid concentration of 100 μM. Thelipid solutions appeared clear after this step, suggesting that bothDLPS and DLEPC either dissolved or formed micellar aggregates insolution. Nevertheless, the lipid suspensions were sonicated for 30 minin a bath sonicator at room temperature to produce small unilamellarliposomes. Upon reconstitution the lipid solutions were clear and wereused without filtering.

Polarized microscopy of the aqueous-liquid crystal interface. Theorientation of TL205 within each optical cell was examined withplane-polarized light in transmission mode on an IX-71 invertedmicroscope with crossed polarizers. The source light intensity levelswere constant for all images of the same magnification. Homeotropicalignments of the liquid crystal were determined by observing theabsence of transmitted light regardless of rotation of the sample.Images were taken with a Hamamatsu camera controlled by ImageProsoftware.

Fluorescent microscopy. Images were obtained with an Olympus IX-71inverted microscope. Fluorescence filter cubes with the appropriatespectral filters for each fluorophore were from Chroma Technology Corp.(Rockingham, Vt.). Images were taken with a Hamamatsu digital cameracontrolled by ImagePro software. The fluorescent images were taken withthe liquid crystal/OTS glass interface toward the objective.

Example 1 Liquid Crystals Report the Presence and Absence of a ProteinLayer in Cell Media

Matrigel adsorbed to solid substrates is known to promote the adhesionand growth of human embryonic stem cells. Consequently, the inventorsfirst sought to determine if it was possible to deposit Matrigel at theaqueous-TL205 interface and to determine the extent to which theorientational order in the liquid crystal was coupled to the presence ofMatrigel. A solution of Matrigel (0.25 mg/mL, 0.1 M sodium bicarbonate)conjugated to the epifluorescent Alexa Fluor 430 label was introducedover a film of TL205 hosted in a gold grid on an OTS-coated glass slideand the Matrigel solution was allowed to gel for 1.5 h at 37° C. Afterexchanging the Matrigel solution with phosphate-buffered saline,fluorescence microscopy was used to image the Matrigel. Fluorescenceimages of Matrigel supported on TL205 that had been subjected to theabove procedure showed areas of intense fluorescence on a uniformlybright background within each grid square (FIG. 1C). The speckledappearance was similar to that observed for Matrigel deposited under thesame conditions on glass controls. This appearance supports theconclusion that Matrigel adsorbs at the aqueous-TL205 interface—aprerequisite for seeding of ES cells at the TL205 surface (see below).

In an effort to estimate the thickness of the Matrigel layer at theaqueous-TL205 interface, the inventors used ellipsometry to measure thethickness of Matrigel films prepared on silicon substrates treated withhydrophobic, cationic, and anionic self-assembled monolayers. In allcases, the ellipsometric thickness of nominally dry Matrigel layers wasmeasured to be ˜10 nm, suggesting that the hydrated Matrigel layers onTL205 are likely on the order of hundreds of nanometers in thickness.

After establishing the presence of Matrigel at the aqueous-TL205interface, polarized light microscopy was used to characterize theorientational order of TL205 on which Matrigel had been deposited usingthe procedure described above. As a reference, the inventors examinedthe optical appearance of TL205 immersed in the buffer used for Matrigeldeposition (no lipids, proteins, or cells present) and observed a brightoptical texture with pale yellow interference colors (FIG. 1D). Thisappearance can be understood as follows: At the TL205-glass interface,the orientation of the liquid crystal is anchored perpendicular to theglass by the monolayer of octadecyltrichlorosilane (shown schematicallyin the director profile in FIG. 1A). Consequently, the bright opticalappearance is the result of in-plane birefringence of the liquid crystalaligned parallel to the aqueous-TL205 interface. The presence of darkbrushes emanating from points along the edges or near the center of eachcompartment indicates that there is a variation of the azimuthal(radial) orientation of the liquid crystal within each compartment ofthe grid.

The optical appearance of TL205 coated with a thin film of Matrigel atthe aqueous-TL205 interface was substantially textured (FIG. 1E) ascompared to the control (FIG. 1D). This appearance is distinct from theoptical appearances of liquid crystals in contact with solutions ofglobular proteins. For example, contact of TL205 with a solution of theglobular protein bovine serum albumin (BSA) yielded an optical texturethat was essentially the same as that exhibited by TL205 in contact withbuffer (FIG. 1F). BSA is a globular protein that likely has littlelong-range organization at the aqueous-TL205 interface, while Matrigelis composed primarily of laminin and collagen fibrils (˜75 nm[23, 25])and ˜300 nm[26] in length, respectively) that are organized into anextended fibrous network.[26] These results suggest that the fibrillarorganization of the Matrigel is reflected in the ordering of the liquidcrystal TL205. This observation also suggests that the liquid crystal isable to report reorganization of Matrigel and other extracellularcomponents by cells.

Example 2 Liquid Crystals Coated with a Protein Layer Support CellGrowth

Next, it was determined if it was possible to seed and grow ES cells onaqueous-TL205 interfaces that had been coated with a Matrigel film. EScells grown to confluence were dissociated from their culture plate andadded to wells containing either TL205 hosted in a gold grid or bareglass (control) at a concentration of 2.5-5.0×105 cells per well. EScells did attach and grow at Matrigel-decorated interfaces of the liquidcrystal (FIG. 2). The development of the ES cell colonies was followedfor up to fourteen days with phase contrast microscopy (FIG. 2A-D).After cell seeding, small colonies of ES cells were observed on theliquid crystal (FIG. 2B). Many of the colonies grew in size over thecourse of seven days (FIG. 2C-D), and occasionally grew to nearconfluence after 14 days. When ES cells were seeded directly on TL205,without a Matrigel coating, no colony attachment or growth was observed.A qualitative calcein-AM based cell viability assay indicated that themajority of stem cells on Matrigel-coated TL205 were alive after four,seven, and 12 days. Oct-4 staining, which is specific for a protein inES cells, showed no qualitative difference in the differentiation of EScells cultured on Matrigel-coated TL205 compared to those cultured onglass controls. Flow cytometry supported the results of both the cellviability and differentiation assays. This corresponds with qualitativedata obtained by Oct-4 staining of stem cells, which indicated cells hadnot differentiated to a higher degree than control wells grown understandard conditions. A lower density of both initial cell colonies andthe rate of radial growth of the colonies on TL205 was observed relativeto controls grown on Matrigel-coated glass. Taken together, theseresults indicate that ES cells are able to attach to Matrigel-coatedTL205 interfaces, survive for days on the TL205 interfaces, in manycases growing into large colonies similar to the colonies. The fluidnature of the underlying TL205 substrate does not appear to inhibit cellattachment or growth.

In parallel to observing the growth of ES cell colonies withphase-contrast imaging, the orientational order within the liquidcrystal was followed by using polarized microscopy (in transmissionmode, illuminated with white light). Changes in the orientational orderof the liquid crystal as ES cell colonies attached and grew at theMatrigel-coated TL205 interface were observed (FIG. 2E-H). Initially,the Matrigel-coated TL205 appeared bright (planar anchoring atinterface) with textures similar to those noted in the discussion above(FIG. 2E). After culturing stem cells for one day on Matrigel-coatedTL205, the appearance of dark regions in the TL205 were observed,indicative of homeotropic anchoring of the liquid crystal. The darkregions generally appeared adjacent to the locations of ES cell colonies(compare FIGS. 2B and 2F) and grew in size as the cell colonies grew(FIG. 2G, H).

Because results suggested that the orientations of TL205 are coupled tothe presence of the Matrigel deposited at the interface (FIG. 1C, 1E),it was determined if the development of regions of homeotropic anchoringwas the result of reorganization of the Matrigel film by the cells.Alexa Fluor 430-labeled Matrigel was used to track the Matrigel locationwith fluorescence microscopy over the course of the ES cell culture(FIG. 2I-L). Initially, a speckled fluorescence on a uniformly brightbackground was observed (as described above), indicating a uniformcoverage of the Matrigel on the liquid crystal (FIG. 2I). Followingseeding of ES cells, the Matrigel fluorescence became non-uniform (FIG.2J-L) and exhibited three distinct levels of fluorescence intensity:regions of fluorescence similar to that observed in the absence ofcells, regions with increased fluorescence intensity, and regions withessentially no fluorescence. A comparison of the fluorescence imageswith the polarized light micrographs revealed that regions of depletedfluorescence intensity—indicative of the removal of Matrigel from theaqueous-TL205 interface—corresponded to regions of homeotropic anchoringof the TL205 (e.g., FIGS. 2J and 2F). A comparison of fluorescence andphase-contrast images showed that regions of increased fluorescenceintensity corresponded to the locations of ES cell colonies (e.g., FIGS.2J and 2B). This was particularly true at short times (1 day ofculture). At longer times, the bright fluorescence typically wasco-located with only portions of the large cell colonies (FIG. 2D, 2L).

These results suggest that the ES cells are exerting a force on theMatrigel film that causes the film to be removed from the areas adjacentto cell colonies. ES cell colonies also appear to condense the Matrigelto areas beneath (or, perhaps, within) the colonies. Significantly, theappearance of the liquid crystal reflects this process as a change frombright to dark (planar to homeotropic) when the Matrigel layer isremoved by the cells.

The cause of the homeotropic anchoring observed when Matrigel had beenremoved from the aqueous-TL205 interface was next determined. It wastested whether components of the cell medium were adsorbing to theaqueous-TL205 interface by exposing TL205 with and without a Matrigelcoating to cell media for four days. The media was changed daily tosimulate cell culture conditions. Following this procedure, nosignificant change in the optical appearance of TL205 coated with aMatrigel film was observed (FIG. 3A; compare to FIG. 1D). In contrast,TL205 lacking a Matrigel film at the aqueous-TL205 interface developeddark domains, corresponding to homeotropic anchoring of TL205 (FIG. 3B).The homeotropic domains were separated by irregular bright regions thatcorresponded to planar anchoring. The optical appearance is reminiscentof the anchoring of the liquid crystal 4′-pentyl-4-cyanobiphenyl (5CB)caused by phospholipids adsorbed at aqueous-5CB interfaces. To confirmthat phospholipids adsorbed at aqueous-TL205 interfaces cause similaranchoring of the TL205, TL205 was exposed to an aqueous suspension ofsmall unilamellar vesicles of 1,2-dilauroyl-sn-glycero-3-phosphocholine(DLPC). The adsorption of DLPC to the aqueous-TL205 interface led to achange in the optical appearance of TL205 from bright (planar anchoring,similar to FIG. 1B) to dark (homeotropic anchoring, FIG. 3C) within 1 h.This result is similar to that observed for the adsorption of DLPC toaqueous-5CB interfaces.

Finally, TL205 was exposed to aqueous solutions of 100 μM DLPC mixedwith 1 μM BSA in an attempt to mimic the mixed nature of the cellculture medium. Initially, TL205 exhibited a bright, planar opticalappearance in crossed polars. After 30 min, the optical appearance ofTL205 was primarily dark (homeotropic anchoring) with thin brightregions (planar anchoring, FIG. 3D). This appearance was reminiscent ofthat observed for TL205 in cell media (FIG. 3B). However, the texturedappearance of TL205 in contact with DLPC and BSA was not stable overtime. After 1 h, the optical appearance of TL205 was completely dark(homeotropic). The fact that the mixed optical texture of TL205 wastransient in DLPC/BSA mixtures, but long-lived (days) in cell medium mayreflect the more complex composition of the cell medium.

These results suggest a mechanism for the connection between theappearance of the liquid crystal and the ES cell colonies growing at theMatrigel-coated TL205 interface: the homeotropic appearance of TL205when Matrigel is removed by the cell colonies may be caused byphospholipids or proteins—perhaps products of cell breakdown—in the cellmedium. The Matrigel layer appears to prevent adsorption of lipids andproteins to the aqueous-TL205 interface, thus maintaining planaranchoring of the TL205. Once the Matrigel is removed from the interfaceby the forces exerted by the ES cell colonies, lipids or proteins fromsolution are free to adsorb to the aqueous-TL205 interface, and cause atransition from planar to homeotropic anchoring of the TL205.

The results set forth herein demonstrate that it is possible to culturehuman embryonic stem cells on interfaces of thermotropic liquid crystalsthat are decorated with thin films of Matrigel. The cells survived forup to 12 days on liquid crystal substrates and showed levels ofdifferentiation comparable to that observed for cells on glass controls.Because the orientational order of the liquid crystal was found to becoupled to the presence of the Matrigel, changes in the orientationalorder of the liquid crystal that can be recorded by using polarizedlight microscopy provides a straightforward way to image thereorganization of Matrigel by the cells. The coupling between theMatrigel and TL205 provides a simple tool for monitoring thereorganization of the Matrigel over time. Accordingly, a new approach tothe culture of cells and measurements of cell-extracellular matrixinteractions is provided by the present invention.

Example 3 Culture of Cells on Polyelectrolyte Multilayer (PEM)-coatedLiquid Crystals

This example demonstrates the culturing of cells on liquid crystals thatare coated with PEM film. The inventors confirmed generation of PEMfilms on a variety of substrates (e.g., silicon chip, glass, and liquidcrystal TL205) by the increased fluorescence signals from the labeledPEM component during film growth. Procedures of PEM deposition on theliquid crystal TL205 are provided below. Additional descriptionregarding the growth of PEMs on liquid crystals can be found in thereferences Lockwood N A, Cadwell K D, Caruso F, et al. “Formation ofpolyelectrolyte multilayer films at interfaces between thermotropicliquid crystals and aqueous phases” Advanced Materials 18 (7): 850, 2006and Tjipto E, Cadwell K D, Quinn J F, et al. “Tailoring the interfacesbetween nematic liquid crystal emulsions and aqueous phases vialayer-by-layer assembly” Nano Letters 6 (10): 2243-2248, 1 2006, allincorporated herein by reference.

Method of Growing PEM

All steps described herein were carried out at room temperature. Allpolymer solutions were prepared at pH 4 (adjusted with HCl) and at 1mg/ml for polyethylenimine (ethoxylated, Mw˜70 k, Sigma, USA) andhyaluronic acid (350 kDa, Genzyme, USA) solutions, and at 0.2 mg/ml forcollagen (Collagen Type I, Sigma, USA) and gelatin (Sigma, USA)solutions. Polyethylenimine was used as a cationic surface charger toprepare a strongly charged surface for adsorption of weakpolyelectrolytes. The ethoxylated side chains of this polyethyeniminemake the polymer highly cationic.

The liquid crystal surface was pre-wetted with acetate buffer at pH 4.Collagen denatures at pH 4. The isoelectric point of hyaluronic acid isaround pH 3. Thus, the PEM is built at pH 4 to preserve the collagenstructure while enhancing the charges carried on the weakpolyelectrolytes. There was allowed 3-5 minutes incubation for theliquid crystal to achieve homogeneous alignment in contact with theaqueous phase. This incubation time also helped the liquid crystal filmstabilize following disturbance from solution addition. All solutionaddition or exchange was carried out with caution as the fluidturbulence could potentially dislodge liquid crystal. Polyethyleniminesolution was then slowly added resulting in the gradual exchange out thebuffer. Incubation was allowed for 10 minutes. The liquid crystalsurface was then rinsed via solution exchange using pH 4 acetate buffer3 times. Hyaluronic acid solution was then added using the methoddescribed for polyethylenimine solution. Incubation was again for 10minutes. Another rinse with pH 4 acetate buffer was performed, asdescribed earlier. Solution exchange was then carried out with collagensolution with subsequent incubation for 30 minutes. Rinses wereperformed before depositing subsequent bilayers of (hyaluronicacid/collagen).

For building PEM with (hylauronic acid/gelatin) bilayers, collagen wassimply substituted with gelatin solution. The procedures aresubstantially similar for building PEM with labeled collagen or gelatin.For building PEM on silicon or glass, the above procedure may bemodified such that the substrates may be immersed (i.e., dipped) in thevarious solutions.

Method for Labeling Collagen or Gelatin with Alexa430 Fluorophor

Collagen or gelatin was dissolved to 1 mg/ml in 0.1M NaHCO₃, pH 8.3 at37 C. Gelatin could be dissolved at higher concentrations. Meanwhile,Alexa 430-succinimidyl ester (Molecular Probes, USA) was diluted in DMSOto 10 mg/ml. While stirring, the diluted Alexa 430 solution was slowlyadded at protein-fluorophore ratio of 10:1 for labeling collagen and20:1 for labeling gelatin. Incubation was at room temperature on arocking platform for 2 hr. Labeled protein was purified by running thelabeling reaction solution through Sephadex G-25 sizing column at roomtemperature. Proteins were eluted with pH 4 acetate buffer. Abicinchoninic acid (BCA) assay was performed to determine labeledprotein concentration.

Method of NIH 3T3 Fibroblast Cell Labeling

Cell labeling was performed using freshly trypsinized cells frompolystyrene culture plates. Cell suspensions of 10⁵ cells/ml wereprepared in culture medium. While stirring, 0.5 vol % of DiD lipophilictracer (Molecular Probes, USA) solution was added and incubationfollowed at 37 C for 15 minutes. The solution was then centrifuged at1500 rpm for 5 minutes and cells resuspended in culture medium. The washprocedure was repeated twice.

Method of Culturing NIH 3T3 Cells on PEM-layered TL205 Liquid CrystalSurface.

Labeled 3T3 cells were utilized in this example to facilitateobservation of cells on the above-described substrates. In practice,standard 8-well chamber slides were used and seeding density of 10³-10⁴cells per well was found optimal. Half of the culturing medium wasexchanged every three days.

FIG. 4 depicts DiD-labeled 3T3 cells on polyethylenimine/(hyaluronicacid/collagen)₅-TL205. The two panels represent pictures acquired fromdifferent wells in a standard 8-well chamber slide at 10 hourspost-seeding. These results demonstrate that it is possible to cultureand maintain cells on PEM coated liquid crystalline substrate.

Example 4 Culturing of Cells on Liquid Crystal Elastomer (LCE)Substrates

This example illustrates the culturing of cells on liquid crystalelastomer substrates. The LCE utilized in this example contained themesogen M4OCH3 and polymethylhydrosiloxane, as described in thereference A. Komp and coworkers “A versatile preparation route for thinfree standing liquid single crystal elastomers” Macromol. Rapid Commun,26: 813-818, 2005, incorporated herein by reference.

It is preferred that the LCE sample be placed into a cell culturingcompartment before any treatment is initiated to avoid future handlingof the sample that might damage the sample. In this example, an LCEsample of size 1 mm×2 mm on octyltrichlorosilane (OTS)-coated glass wasplaced in an 8-well chamber slide. LCE was first sterilized with ethanoland then treated by soaking it in bovine calf serum for 5 days withserum exchanged every day. LCE was then conditioned in phosphatebuffered saline (PBS) for 10 minutes, followed by incubation in 10 μg/mlfibronectin (Invitrogen, USA) solution at 4 C overnight. Thefibronectin-coated LCE was then rinsed with PBS twice prior to cellseeding. For the 8-well chamber slide, it is preferred to exchange themedium every three days.

Results of Cell Survival and Proliferation on LCE.

Cell growth was observed on both glass (control) and on LCE. FIG. 5illustrates that the two materials were comparable in terms of celldensity at 12 hour post-cell seeding. NIH 3T3 cell grown on OTS-coatedglass are shown in a phase contrast image in the left panel of FIG. 5.NIH 3T3 cells grown on the above-described LCE are shown the fluorescentimage (right panel) of labeled cells in a well treated with bovine calfserum.

Fibroblasts typically require 4-6 hrs to fully spread on polystyreneculture plates following standard passage protocols. A twelve hourwindow for cell attachment allowed sufficient time for cells to spreadand grow on LCE.

FIG. 6 provides an image of cells on serum-treated LCE on the 5^(th) daypost-cell seeding. The sample shown is that same LCE substrate surfacethat was shown in the comparative FIG. 5.

The results provided in this example demonstrate that cells attach,spread and proliferate on LCE substrates.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thespecific materials and methods described herein. Such equivalents areconsidered to be within the scope of this invention and encompassed bythe following claims.

1. A method of culturing cells on a cell culture substrate, comprising: (a) providing a cell substrate by: (i) preparing an interface between an aqueous phase and a liquid crystalline phase; and (ii) depositing a protein or peptide layer at said interface, wherein said protein or peptide layer comprises at least one protein or peptide that is an extracellular matrix component; (b) seeding living cells onto said protein or peptide layer; and (c) culturing said living cells on the cell culture substrate.
 2. The method according to claim 1 wherein the protein or peptide layer comprises gelatin, laminin, collagen, elastin, or mixtures thereof.
 3. The method according to claim 1 wherein said protein or peptide layer comprises a synthetic polypeptide.
 4. The method according to claim 1 wherein the liquid crystalline phase is a liquid crystal elastomer.
 5. The method according to claim 1, further including a step of forming a polyelectrolyte multilayer (PEM) film at the interface between said aqueous phase and liquid crystalline phase wherein said protein or peptide layer comprises the PEM film.
 6. The method according to claim 1 wherein said living cells are human embryonic stem cells. 